Heat resistant plants and plant tissues and methods and materials for making and using same

ABSTRACT

The subject invention concerns materials and methods for providing plants or plant tissue with increased resistance to heat conditions and/or increased starch biosynthesis. Increased resistance of a plant or plant tissue to heat conditions provides for decreased yield losses as compared to the yield losses generally observed at elevated temperatures. One aspect of the invention concerns polynucleotides that encode a mutant plant small subunit of AGPase. The subject invention also comprises a mutant plant small subunit of AGPase encoded by a polynucleotide of the invention. The subject invention also concerns plants comprising a polynucleotide of the invention and method for making the plants.

GOVERNMENT SUPPORT

The subject matter of this application has been supported by a researchgrant from the National Science Foundation under grant numberIOS-0444031. Accordingly, the government has certain rights in thisinvention.

BACKGROUND OF THE INVENTION

Heat stress leads to decreased maize yield (Peters et al., 1971;Thompson, 1975; Chang, 1981; Christy and Williamson, 1985). This can beattributed to reduced photosynthate availability and transportation fromsource to sink tissues, poor pollination, reduced cell and granule sizeand number, early seed abortion and/or reduced grain filling period.Growth of endosperm starts with a lag phase in which cells activelydivide and continues with a linear phase in which cells increase in sizeand starch synthesis occurs. Elevated temperature during lag phaseresulted in reduced yield (Jones et al., 1984). These investigatorssuggested that reduced yield was due to reduced cell and granule numberand size as well as seed abortion. Additionally, elevated temperaturesduring the linear phase resulted in shorter grain filling period andsubsequently smaller kernels (Jones et al., 1984). Similar results werefound by Hunter et al. (1977) and Tollenaar and Bruulsema (1988).

Records from five states that traditionally produce more than 50% of theUS corn showed that average daily temperature was 23.6° C., around 2° C.higher than optimum during grain filling (Singletary et al., 1994).Photosynthate availability during grain filling is not reduced at hightemperatures, at least in barley and wheat. Indeed, sucrose content inbarley and wheat seeds was either unchanged or elevated at hightemperatures (Bhullar and Jenner, 1986; Wallwork et al., 1998). Alsophotosynthesis in maize increases up to 32° C. (Duncan and Hesketh,1968; Hofstra and Hesketh, 1969; Christy et al., 1985). Moreover,Cheikhn and Jones (1995) studied the ability of maize kernels to fix ¹⁴Csucrose and hexoses at different temperatures. They found that thesesugars increased in the seed at elevated-temperatures. The evidenceabove suggests that limited sugar availability and transport into thekernel during grain filling are not the cause of temperature-inducedyield decreases.

There have been extensive efforts to identify biochemical pathways thatimpact grain filling during elevated temperatures. Singletary et al.(1993; 1994) assayed starch biosynthetic enzymes in maize kernels grownin vitro at elevated temperatures (22° C. to 36° C.). They found thatADP-glucose pyrophosphorylase (AGPase) and soluble starch synthase (SSS)were more heat labile compared to other enzymes participating in starchsynthesis. They suggested that heat lability of AGPase and SSScontributes to grain filling cessation. Duke and Doehlert (1996) foundthat transcripts of several genes encoding enzymes of the starchsynthesis pathway, including those encoding AGPase, were decreased at35° C. compared to 25° C. However, enzyme assays showed that only AGPaseactivity was strikingly lower. They suggested that this could be due toa higher turnover rate of AGPase compared to other enzymes. Finally,Wilhelm et al. (1999), through Q₁₀ analysis, showed that AGPase had themost pronounced reduction in activity compared to several other enzymes.Maize AGPase indeed lost 96% of its activity when heated at 57° C. for 5min (Hannah et al., 1980).

AGPase catalyzes the first committed step in starch (plants) andglycogen (bacteria) synthesis. It involves the conversion of glucose-1-P(G-1-P) and ATP to ADP-glucose and pyrophosphate (PPi). AGPase is aheterotetramer in plants consisting of two identical small and twoidentical large subunits. The large and the small subunits are encodedby shrunken-2 (Sh2) and brittle-2 (Bt2) respectively in maize endosperm.AGPase is allosterically regulated by small effector molecules that areindicative of the energy status of the cell. AGPase is activated by3-PGA, the first carbon assimilatory product, and inhibited/deactivatedby inorganic phosphate (Pi) in cyanobacteria, green algae andangiosperms.

The importance of maize endosperm AGPase in starch synthesis has beenshown by the kernel phenotype of mutants in either subunit of theenzyme. Indeed, such mutants result in shrunken kernels and a largereduction in endosperm starch content (Tsai and Nelson, 1966; Hannah andNelson, 1976). There is also evidence that AGPase catalyses arate-limiting step in starch synthesis (Stark et al. 1992; Giroux et al.1996; Greene et al. 1998; Sakulsingharoja et al. 2004; Obana et al.2006; Wang et al. 2007).

Greene and Hannah (1998a) isolated a mutant form of maize AGPase with asingle amino acid change in the large subunit termed HS33. They showedthat the altered enzyme was more heat-stable and that stability was dueto stronger subunit-subunit interactions. When wheat and rice weretransformed with a Sh2 variant that contains the HS33 change along witha change that affects the allosteric properties of AGPase (Rev6) (Girouxet al., 1996), yield was increased by 38% and 23% respectively(Smidansky et al., 2002; 2003). Remarkably, the increase was due to anincrease in seed number rather than individual seed weight.

Transformation of maize with the Sh2 variant containing the Rev6 andHS33 changes also gives rise to enhanced seed number. Seed yield/ear canbe increased up to 68% in maize. A detailed characterization of themaize transgenic events is under way (Greene and Hannah, inpreparation). Enhanced seed number cannot be explained by Rev6 since,when expressed alone in maize, it increases only seed weight (Hannah,unpublished). The above studies show the importance of AGPase heatstability in cereal yield.

Cross et al. (2004) generated a mosaic small subunit (MP) consisting ofthe first 200 amino acids of BT2 and the last 275 amino acids of thepotato tuber small subunit. MP in a complex with SH2 had severalfeatures that could lead to agronomic gain (Cross et al., 2004; Boehleinet al., 2005). Some of those features were increased activity in theabsence of the activator 3-PGA, increased affinity for 3-PGA andelevated heat stability compared to wildtype maize endosperm AGPase(BT2/SH2). Preliminary data show that maize plants with transgenic MPcontaining AGPase variant expressed in maize endosperm provides for astarch yield increase (Hannah, unpublished data).

BRIEF SUMMARY OF THE INVENTION

The subject invention concerns materials and methods for providingplants or plant tissue with increased resistance to heat conditionsand/or increased starch biosynthesis. Increased resistance of a plant orplant tissue to heat conditions provides for decreased yield losses ascompared to the yield losses generally observed at elevatedtemperatures. One aspect of the invention concerns polynucleotides thatencode a mutant plant small subunit of AGPase. In one embodiment, apolynucleotide of the invention encodes a plant AGPase small subunithaving an amino acid mutation wherein the threonine amino acidcorresponding to amino acid position 462 of wild type maize AGPase smallsubunit is substituted with an amino acid that confers increased heatstability. In another embodiment, a polynucleotide encodes a chimericplant AGPase small subunit composed of sequences from two differentplants (as described in U.S. Pat. No. 7,173,165) and comprising an aminoacid mutation of the invention wherein the threonine amino acidcorresponding to amino acid position 462 of wild type maize AGPase smallsubunit is substituted with an amino acid that confers increased heatstability. The mutation in the chimeric AGPase synergistically enhancesheat stability. The subject invention also comprises a mutant plantsmall subunit of AGPase encoded by a polynucleotide of the invention.Characterization of heat stability as well as kinetic and allostericproperties indicates increased starch yield is provided when thepolynucleotides of the invention are expressed in plants such as monocotendosperms.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows glycogen produced by E. coli cells expressing BT2, BT2-TI,MP, MP-TI along with SH2. Glycogen from cells expressing only SH2, BT2,BT2-TI, and MP alone. Glycogen is measured in glucose units. The errorbars indicate standard deviation (N=3).

FIGS. 2A and 2B show dot blots of crude extracts from E. coli cellsexpressing BT2, BT2-TI, MP, MP-TI with the complementary subunit SH2.AGPase was visualized by using a monoclonal antibody against BT2. Thedensity of the spots was estimated by using ImageJ(http://rsb.info.nih.gov/ij/).

FIG. 3 shows specific activity of AGPase variants in crude and partiallypurified protein extracts from non-induced E. coli cells. Activity wasmeasured in the reverse direction. n.d.=not detectable. The error barsindicate standard deviation (N=3).

FIG. 4 shows purification of AGPase. SDS-PAGE of purified recombinantBT2/SH2, TI/SH2, MP/SH2, and MP-TI/SH2. Precision Plus Protein All BlueStandard from Biorad was used as a marker. The upper arrow on the leftpoints to the large subunit. The lower arrow on the left points to thesmall subunit.

FIGS. 5A and 5B show heat stability of purified BT2/SH2, BT2-TI/SH2, andMP/SH2. The half-life (T_(1/2)) of each AGPase is expressed asmean±standard error. The p-values are estimated by an F-test implementedby Prizm (Graph pad, San Diego Calif.). In FIG. 5A, the assay wasconducted in the forward direction. In FIG. 5B, the assay was conductedin the reverse direction.

FIGS. 6A and 6B show heat stability of purified MP/SH2 and MP-TI/SH2.The half-life (T_(1/2)) of each AGPase is expressed as mean±standarderror. The p-values are estimated by an F-test implemented by Prizm(Graph pad, San Diego Calif.). In FIG. 6A, the assay was conducted inthe forward direction. In FIG. 6B, the assay was conducted in thereverse direction.

FIGS. 7A-7C show 3D modeling of BT2 and TI. FIG. 7A is the predicted 3Dstructure of BT2 monomer. The TI change is marked by a red circle. Theareas of BT2 that are directly involved in subunit-subunit interactionsare highlighted by yellow boxes. FIG. 7B shows the distances of carbonatoms of Thr462 (1,2) from those of Pro60 (4,5) and Leu61 (3). FIG. 7Cshows the distances of carbon atoms of Ile462 (1,2,3,4) from those ofPro60 (5,6) and Leu61 (7,8). The Thr462 and Ile462 contacting residueswere determined by using FirstGlance Jmol. Dark gray spheres indicatecarbon atoms of Thr462 and Ile462. Light gray spheres indicate carbonatoms of contacting residues. Oxygen and nitrogen atoms are indicated byred and blue color respectively.

FIG. 8 show strength of AGPase subunit-subunit interactions. SH2 wasused as a bait and BT2, TI, and MP as a prey in a yeast two hybridsystem. A quantitative β-galactosidase assay was used to quantify theinteractions between the bait and the prey. The error bars indicate 2×standard error (N=4).

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO:1 is a polynucleotide sequence comprising a nucleotidesequence encoding a mutant polypeptide (TI) of the invention.

SEQ ID NO:2 is an amino acid sequence of a mutant polypeptide (TI) ofthe invention.

SEQ ID NO:3 is a polynucleotide sequence comprising a nucleotidesequence encoding a mutant polypeptide (MP-TI) of the invention.

SEQ ID NO:4 is an amino acid sequence of a mutant polypeptide (MP-TI) ofthe invention.

SEQ ID NO:5 is an amino acid sequence of a mutant polypeptide (TI+YC) ofthe invention.

SEQ ID NO:6 is an amino acid sequence of a mutant polypeptide (TI+QTCL)of the invention.

SEQ ID NO:7 is an amino acid sequence of a mutant polypeptide (TI+ETCL)of the invention.

SEQ ID NO:8 is an amino acid sequence of a mutant polypeptide (MP-TI+YC)of the invention.

SEQ ID NO:9 is an amino acid sequence of a mutant polypeptide(MP-TI+QTCL) of the invention.

SEQ ID NO:10 is an amino acid sequence of a mutant polypeptide(MP-TI+ETCL) of the invention.

SEQ ID NO:11 is a polynucleotide sequence comprising a nucleotidesequence encoding a mutant polypeptide (SEQ ID NO:5) of the invention.

SEQ ID NO:12 is a polynucleotide sequence comprising a nucleotidesequence encoding a mutant polypeptide (SEQ ID NO:6) of the invention.

SEQ ID NO:13 is a polynucleotide sequence comprising a nucleotidesequence encoding a mutant polypeptide (SEQ ID NO:7) of the invention.

SEQ ID NO:14 is a polynucleotide sequence comprising a nucleotidesequence encoding a mutant polypeptide (SEQ ID NO:8) of the invention.

SEQ ID NO:15 is a polynucleotide sequence comprising a nucleotidesequence encoding a mutant polypeptide (SEQ ID NO:9) of the invention.

SEQ ID NO:16 is a polynucleotide sequence comprising a nucleotidesequence encoding a mutant polypeptide (SEQ ID NO:10) of the invention.

SEQ ID NO:17 is an oligonucleotide that can be used according to thepresent invention.

SEQ ID NO:18 is an oligonucleotide that can be used according to thepresent invention.

SEQ ID NO:19 is an oligonucleotide that can be used according to thepresent invention.

SEQ ID NO:20 is an oligonucleotide that can be used according to thepresent invention.

SEQ ID NO:21 is an amino acid sequence of a mutant polypeptide (TI) ofthe invention.

SEQ ID NO:22 is an amino acid sequence of a mutant polypeptide (TI) ofthe invention.

SEQ ID NO:23 is an amino acid sequence of a mutant polypeptide (TI) ofthe invention.

SEQ ID NO:24 is an amino acid sequence of a mutant polypeptide (TI) ofthe invention.

SEQ ID NO:25 is an amino acid sequence of a mutant polypeptide (TI) ofthe invention.

SEQ ID NO:26 is an amino acid sequence of potato tuber AGPase smallsubunit protein.

DETAILED DESCRIPTION OF THE INVENTION

The subject invention concerns materials and methods for providingplants with increased resistance to heat conditions and/or increasedstarch biosynthesis. Increased resistance of a plant to heat conditionsprovides for decreased yield losses as compared to yield lossesgenerally observed at elevated temperatures.

One aspect of the invention concerns polynucleotides that encode amutant plant small subunit of AGPase. In one embodiment, apolynucleotide of the invention encodes a plant AGPase small subunithaving an amino acid mutation wherein the threonine amino acidcorresponding to amino acid position 462 of wild type maize endospermAGPase small subunit is substituted with an amino acid that confersincreased heat stability. In a specific embodiment, the amino acidsubstituted is an isoleucine. In one embodiment, the mutant plant AGPasesmall subunit is maize endosperm AGPase small subunit. In an exemplifiedembodiment, the mutant plant AGPase small subunit comprises the aminoacid sequence shown in SEQ ID NO:2, or a fragment or variant thereof. Ina specific embodiment, the polynucleotide comprises the nucleotidesequence shown in SEQ ID NO:1, or a fragment or variant thereof. Inanother embodiment, the mutant plant AGPase small subunit is barley,wheat, sorghum, potato, or rice. In a specific embodiment, the mutantbarley, wheat, sorghum, or potato AGPase small subunit comprises theamino acid sequence shown in SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23,SEQ ID NO:24, or SEQ ID NO:25, respectively. In another embodiment, thepolynucleotide encodes a mutant plant AGPase small subunit that canadditionally comprise an amino acid mutation as described in publishedInternational patent application WO 2005/019425 (Hannah and Linebarger).In one embodiment, the mutant AGPase small subunit encoded by thepolynucleotide comprises an amino acid mutation wherein the tyrosinecorresponding to amino acid position 36 of wild type maize endospermAGPase is substituted with a cysteine. The mutant AGPase small subunitcan also optionally comprise an amino acid inserted between the serineand threonine amino acids corresponding to amino acid positions 34 and35 of wild type maize endosperm AGPase, respectively. In specificembodiments, the amino acid inserted between amino acids at position 34and 35 of the AGPase small subunit is a glutamic acid or glutamine. Inexemplified embodiments, the mutant plant AGPase small subunit comprisesthe amino acid sequence shown in SEQ ID NO:5, SEQ ID NO:6, or SEQ IDNO:7, or a fragment or variant thereof. In specific embodiments, thepolynucleotide comprises the nucleotide sequences shown in SEQ ID NO:11,SEQ ID NO:12, or SEQ ID NO:13, or a fragment or variant thereof.

In another embodiment, a polynucleotide encodes a chimeric plant AGPasesmall subunit composed of sequences from two different plants (asdescribed in U.S. Pat. No. 7,173,165) and also comprising an amino acidmutation of the invention wherein the threonine amino acid correspondingto amino acid position 462 of wild type maize endosperm AGPase smallsubunit is substituted with an amino acid that confers increased heatstability. In a specific embodiment, the amino acid substituted is anisoleucine. In one embodiment, the chimeric AGPase small subunitcomprises a C-terminal portion from one plant and an N-terminal portionfrom another plant. In one embodiment, a chimeric protein of the presentinvention comprises an N-terminus sequence having approximately thefirst 150 to 250 amino acids of the N-terminus of a first plant AGPasesmall subunit and a C-terminus sequence comprising approximately theterminal 300 residues or less of the C-terminus of a second plant AGPasesmall subunit. Thus, the C-terminus of the chimeric subunit can comprisethe terminal 300, or 299, or 298, or 297, or 296, or 295, and so forth,residues of the C-terminus of the second plant. The subunit sequencescan be from an AGPase of a monocot or dicot plant, or both a monocot anda dicot. Monocotyledonous plants, such as, for example, rice, wheat,barley, oats, sorghum, maize, lilies, and millet are included within thescope of the invention. Dicot plants can include, for example, tobacco,soybean, potato, sweet potato, radish, cabbage, rape, apple tree, andlettuce. In one embodiment, the first 200 or so amino acids of theN-terminus of the chimeric protein are from the N-terminus of maizeendosperm AGPase small subunit and the C-terminus amino acids are fromthe C-terminus of potato tuber AGPase small subunit and include themutation corresponding to amino acid position 462 of the presentinvention. In a specific embodiment, the C-terminus region of a chimericprotein of the present invention comprises the terminal 276 amino acidsof the AGPase small subunit of potato tuber. In an exemplifiedembodiment, the chimeric protein comprises a portion of the smallsubunit of maize endosperm AGPase and a portion of the small subunit ofpotato tuber AGPase. In a specific embodiment, the chimeric proteincontains a) the first 199 amino acids (i.e., amino acids 1 through 199)from the small subunit of maize endosperm AGPase and the carboxylterminal end of the small subunit of potato tuber AGPase, starting atamino acid 246 (i.e., amino acids 246 through 521) using the amino acidsequence shown for the protein deposited as Genbank accession numberX61186 (or, alternatively, starting at amino acid 175 using thenumbering system for the potato AGPase subunit as in Hannah et al.,2001) and b) the mutation corresponding to amino acid position 462 ofthe present invention. In an exemplified embodiment, the plant chimericAGPase small subunit comprises the amino acid sequence shown in SEQ IDNO:4, or a fragment or variant thereof. In a specific embodiment, thepolynucleotide comprises the nucleotide sequence shown in SEQ ID NO:3,or a fragment or variant thereof. In another embodiment, thepolynucleotide encodes a mutant plant AGPase small subunit that canadditionally comprise an amino acid mutation described in publishedInternational patent application WO 2005/019425 (Hannah and Linebarger).In a further embodiment, the mutant AGPase small subunit encoded by thepolynucleotide also comprises an amino acid mutation wherein a tyrosineat position 36 is substituted with a cysteine. The mutant AGPase smallsubunit can also optionally comprise an amino acid inserted between theserine and threonine amino acids at positions 34 and 35, respectively.In specific embodiments, the amino acid inserted between position 34 and35 of the mutant AGPase small subunit is a glutamic acid or glutamine.In exemplified embodiments, the mutant plant AGPase small subunitcomprises the amino acid sequence shown in SEQ ID NO:8, SEQ ID NO:9, orSEQ ID NO:10, or a fragment or variant thereof. In specific embodiments,the polynucleotide comprises the nucleotide sequences shown in SEQ IDNO:14, SEQ ID NO:15, or SEQ ID NO:16, or a fragment or variant thereof.

The subject invention also concerns methods for increasing heatstability and/or increasing starch biosynthesis, and increasing cropyield of a plant or plant tissue. In one embodiment, a method of theinvention comprises introducing one or more polynucleotides of thepresent invention into a plant. In certain embodiments, thepolynucleotides introduced into the plant encode one or morepolypeptides comprising the amino acid sequence shown in any of SEQ IDNO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8,SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQID NO:24, or SEQ ID NO:25, or a fragment or variant thereof. In aspecific embodiment, the polynucleotide comprises the nucleotidesequence shown in SEQ ID NO:1, or SEQ ID NO:3, or a fragment or variantthereof. In further specific embodiments, the polynucleotide comprisesthe nucleotide sequences shown in SEQ ID NO:11, SEQ ID NO:12, SEQ IDNO:13, SEQ ID NO:14, SEQ ID NO:15, or SEQ ID NO:16, or a fragment orvariant thereof. In one embodiment, the polynucleotide is stablyincorporated into the genome of the plant or plant tissue. Thepolynucleotide can comprise regulatory elements, such as a promoterand/or enhancer sequences, that provide for increased expression of thepolynucleotide and/or the polypeptide encoded thereby. In a specificembodiment, the promoter sequence is one that provides for constitutiveor tissue-specific (e.g., endosperm) expression. Plants or plant tissuescontaining the polynucleotide, or progeny of the plants, optionally canbe screened for increased expression of a polynucleotide or polypeptideof the invention. In one embodiment, multiple copies of one or morepolynucleotides of the invention are introduced into a plant or planttissue and stably incorporated into the genome of the plant. In oneembodiment, a polynucleotide of the invention is provided in anexpression construct as described herein.

The subject invention also concerns mutant AGPase small subunitpolypeptides encoded by the polynucleotides of the invention. In oneembodiment, the polypeptide comprises the amino acid sequence shown inSEQ ID NO:2, or a fragment or variant thereof. In other embodiments, thepolypeptide comprises the amino acid sequence shown in SEQ ID NO:21, SEQID NO:22, SEQ ID NO:23, SEQ ID NO:24, or SEQ ID NO:25, or a fragment orvariant thereof. In another embodiment, the polypeptide comprises theamino acid sequence shown in SEQ ID NO:4, or a fragment or variantthereof. In still a further embodiment, the polypeptide comprises theamino acid sequence shown in any of SEQ ID NO:5, SEQ ID NO:6, SEQ IDNO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:10, or a fragment orvariant thereof.

The subject invention also concerns mutant plant AGPase enzymescomprising one or more mutant AGPase small subunit polypeptides of theinvention. The mutant plant AGPase can also comprise one or more wildtype AGPase large subunit polypeptides. In specific embodiments, amutant plant AGPase enzyme comprises one or more mutant AGPase smallsubunit polypeptides any of which can comprise the amino acid sequenceof SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:21, SEQ ID NO:22, SEQ IDNO:23, SEQ ID NO:24, or SEQ ID NO:25, or a fragment or variant of anysuch sequence, wherein the mutant AGPase enzyme exhibits increased heatstability relative to a wild type AGPase enzyme. In one embodiment, themutant plant enzyme comprises two mutant AGPase small subunits of theinvention, wherein the mutant polypeptides can have the same mutation(s)or can have different mutation(s). The subject invention also concernsmutant plant AGPase enzymes comprising one or more mutant AGPase smallsubunit polypeptides of the invention and one or more mutant AGPaselarge subunit polypeptides. In one embodiment, the mutant AGPase largesubunit polypeptide can be any of those as described in any of U.S. Pat.Nos. 5,589,618; 5,650,557; 5,872,216; 6,069,300; 6,184,438; 6,403,863;6,809,235; 7,173,165; 7,312,378; and 6,969,783. In one embodiment, amutant AGPase large subunit polypeptide comprises a Rev6 mutation. Inanother embodiment, a mutant AGPase large subunit polypeptide comprisesone or more heat stable (HS) mutations, as described in U.S. Pat. Nos.6,069,300; 6,403,863; 6,809,235; 7,312,378; and 6,969,783, and publishedInternational patent application nos. WO 99/58698; WO 2003/0070901; WO98/22601; and WO 02/072784, such as, for example, the HS33 mutation. Inone embodiment, the mutant plant AGPase enzyme comprises two mutantAGPase small subunit polypeptides of the invention, wherein the mutantAGPase small subunit polypeptides can have the same mutation(s) or canhave different mutation(s), as described herein. In another embodiment,the mutant plant AGPase enzyme comprises two mutant AGPase large subunitpolypeptides wherein the mutant AGPase large subunit polypeptides canhave the same mutation(s) or can have different mutation(s). In afurther embodiment, the mutant plant AGPase enzyme comprises two mutantAGPase small subunit polypeptides of the invention and two mutant SH2polypeptides, wherein the mutant AGPase small subunit polypeptides andthe mutant AGPase large subunit polypeptides can have the samemutation(s) or can have different mutation(s), as described herein.

The subject invention also concerns methods for providing for a mutantplant AGPase enzyme having increased heat stability relative to wildtype plant AGPase. In one embodiment, the method comprises incorporatingor providing one or more mutant AGPase small subunit polypeptides of thepresent invention with wild type or mutant AGPase large subunits in anAGPase enzyme. In one embodiment, the AGPase enzyme comprises a tetramerof polypeptide subunits, wherein one, two, or more of the subunits is amutant polypeptide of the present invention. In one embodiment, theAGPase enzyme also comprises a mutant AGPase large subunit polypeptidesubunit, such as a mutant large subunit comprising a Rev6 and/or a heatstability mutation, such as HS33.

The subject invention also concerns plants, plant tissue, and plantcells of the invention that comprise a polynucleotide or the proteinencoded by the polynucleotide of the invention, or that express a mutantpolypeptide of the invention, or a fragment or variant thereof, or thatcomprise or express a mutant plant AGP enzyme of the present invention.Plant tissue includes, but is not limited to, seed, scion, androotstock. Plants within the scope of the present invention includemonocotyledonous plants, such as, for example, rice, wheat, barley,oats, rye, sorghum, maize, sugarcane, pineapple, onion, bananas,coconut, lilies, turfgrasses, and millet. Plants within the scope of thepresent invention also include dicotyledonous plants, such as, forexample, tomato, cucumber, squash, peas, alfalfa, melon, chickpea,chicory, clover, kale, lentil, soybean, beans, tobacco, potato, sweetpotato, yams, cassava, radish, broccoli, spinach, cabbage, rape, appletrees, citrus (including oranges, mandarins, grapefruit, lemons, limesand the like), grape, cotton, sunflower, strawberry, lettuce, and hop.Herb plants containing a polynucleotide of the invention are alsocontemplated within the scope of the invention. Herb plants includeparsley, sage, rosemary, thyme, and the like. In one embodiment, theplant, plant tissue, or plant cell is Zea mays. In one embodiment, aplant, plant tissue, or plant cell is a transgenic plant, plant tissue,or plant cell. In another embodiment, a plant, plant tissue, or plantcell is one that has been obtained through a breeding program.

Polynucleotides useful in the present invention can be provided in anexpression construct. Expression constructs of the invention generallyinclude regulatory elements that are functional in the intended hostcell in which the expression construct is to be expressed. Thus, aperson of ordinary skill in the art can select regulatory elements foruse in bacterial host cells, yeast host cells, plant host cells, insecthost cells, mammalian host cells, and human host cells. Regulatoryelements include promoters, transcription termination sequences,translation termination sequences, enhancers, and polyadenylationelements. As used herein, the term “expression construct” refers to acombination of nucleic acid sequences that provides for transcription ofan operably linked nucleic acid sequence. As used herein, the term“operably linked” refers to a juxtaposition of the components describedwherein the components are in a relationship that permits them tofunction in their intended manner. In general, operably linkedcomponents are in contiguous relation.

An expression construct of the invention can comprise a promotersequence operably linked to a polynucleotide sequence encoding a mutantpolypeptide of the invention. Promoters can be incorporated into apolynucleotide using standard techniques known in the art. Multiplecopies of promoters or multiple promoters can be used in an expressionconstruct of the invention. In a preferred embodiment, a promoter can bepositioned about the same distance from the transcription start site inthe expression construct as it is from the transcription start site inits natural genetic environment. Some variation in this distance ispermitted without substantial decrease in promoter activity. Atranscription start site is typically included in the expressionconstruct.

If the expression construct is to be provided in or introduced into aplant cell, then plant viral promoters, such as, for example, acauliflower mosaic virus (CaMV) 35S (including the enhanced CaMV 35Spromoter (see, for example U.S. Pat. No. 5,106,739)) or a CaMV 19Spromoter or a cassava vein mosaic can be used. Other promoters that canbe used for expression constructs in plants include, for example,prolifera promoter, Ap3 promoter, heat shock promoters, T-DNA 1′- or2′-promoter of A. tumefaciens, polygalacturonase promoter, chalconesynthase A (CHS-A) promoter from petunia, tobacco PR-1a promoter,ubiquitin promoter, actin promoter, alcA gene promoter, pin2 promoter(Xu et al., 1993), maize WipI promoter, maize trpA gene promoter (U.S.Pat. No. 5,625,136), maize CDPK gene promoter, and RUBISCO SSU promoter(U.S. Pat. No. 5,034,322) can also be used. Tissue-specific promoters,for example fruit-specific promoters, such as the E8 promoter of tomato(accession number: AF515784; Good et al. (1994)) can be used.Fruit-specific promoters such as flower organ-specific promoters can beused with an expression construct of the present invention forexpressing a polynucleotide of the invention in the flower organ of aplant. Examples of flower organ-specific promoters include any of thepromoter sequences described in U.S. Pat. Nos. 6,462,185; 5,639,948; and5,589,610. Seed-specific promoters such as the promoter from aβ-phaseolin gene (for example, of kidney bean) or a glycinin gene (forexample, of soybean), and others, can also be used. Endosperm-specificpromoters include, but are not limited to, MEG1 (EPO application No.EP1528104) and those described by Wu et al. (1998), Furtado et al.(2001), and Hwang et al. (2002). Root-specific promoters, such as any ofthe promoter sequences described in U.S. Pat. No. 6,455,760 or U.S. Pat.No. 6,696,623, or in published U.S. patent application Nos. 20040078841;20040067506; 20040019934; 20030177536; 20030084486; or 20040123349, canbe used with an expression construct of the invention. Constitutivepromoters (such as the CaMV, ubiquitin, actin, or NOS promoter),developmentally-regulated promoters, and inducible promoters (such asthose promoters than can be induced by heat, light, hormones, orchemicals) are also contemplated for use with polynucleotide expressionconstructs of the invention.

Expression constructs of the invention may optionally contain atranscription termination sequence, a translation termination sequence,a sequence encoding a signal peptide, and/or enhancer elements.Transcription termination regions can typically be obtained from the 3′untranslated region of a eukaryotic or viral gene sequence.Transcription termination sequences can be positioned downstream of acoding sequence to provide for efficient termination. A signal peptidesequence is a short amino acid sequence typically present at the aminoterminus of a protein that is responsible for the relocation of anoperably linked mature polypeptide to a wide range of post-translationalcellular destinations, ranging from a specific organelle compartment tosites of protein action and the extracellular environment. Targetinggene products to an intended cellular and/or extracellular destinationthrough the use of an operably linked signal peptide sequence iscontemplated for use with the polypeptides of the invention. Classicalenhancers are cis-acting elements that increase gene transcription andcan also be included in the expression construct. Classical enhancerelements are known in the art, and include, but are not limited to, theCaMV 35S enhancer element, cytomegalovirus (CMV) early promoter enhancerelement, and the SV40 enhancer element. Intron-mediated enhancerelements that enhance gene expression are also known in the art. Theseelements must be present within the transcribed region and areorientation dependent. Examples include the maize shrunken-1 enhancerelement (Clancy and Hannah, 2002).

DNA sequences which direct polyadenylation of mRNA transcribed from theexpression construct can also be included in the expression construct,and include, but are not limited to, an octopine synthase or nopalinesynthase signal. The expression constructs of the invention can alsoinclude a polynucleotide sequence that directs transposition of othergenes, i.e., a transposon.

Polynucleotides of the present invention can be composed of either RNAor DNA. Preferably, the polynucleotides are composed of DNA. The subjectinvention also encompasses those polynucleotides that are complementaryin sequence to the polynucleotides disclosed herein. Polynucleotides andpolypeptides of the invention can be provided in purified or isolatedform.

Because of the degeneracy of the genetic code, a variety of differentpolynucleotide sequences can encode mutant polypeptides of the presentinvention. A table showing all possible triplet codons (and where U alsostands for T) and the amino acid encoded by each codon is described inLewin (1985). In addition, it is well within the skill of a persontrained in the art to create alternative polynucleotide sequencesencoding the same, or essentially the same, mutant polypeptides of thesubject invention. These variant or alternative polynucleotide sequencesare within the scope of the subject invention. As used herein,references to “essentially the same” sequence refers to sequences whichencode amino acid substitutions, deletions, additions, or insertionswhich do not materially alter the functional activity of the polypeptideencoded by the polynucleotides of the present invention. Allelicvariants of the nucleotide sequences encoding a wild type or mutantpolypeptide of the invention are also encompassed within the scope ofthe invention.

Substitution of amino acids other than those specifically exemplified ornaturally present in a wild type or mutant polypeptide and/or AGPaseenzyme of the invention are also contemplated within the scope of thepresent invention. For example, non-natural amino acids can besubstituted for the amino acids of a mutant AGPase small subunitpolypeptide, so long as the mutant polypeptide having the substitutedamino acids retains substantially the same functional activity as themutant polypeptide in which amino acids have not been substituted.Examples of non-natural amino acids include, but are not limited to,ornithine, citrulline, hydroxyproline, homoserine, phenylglycine,taurine, iodotyrosine, 2,4-diaminobutyric acid, a-amino isobutyric acid,4-aminobutyric acid, 2-amino butyric acid, γ-amino butyric acid, e-aminohexanoic acid, 6-amino hexanoic acid, 2-amino isobutyric acid, 3-aminopropionic acid, norleucine, norvaline, sarcosine, homocitrulline,cysteic acid, t-butylglycine, t-butylalanine, phenylglycine,cyclohexylalanine, β-alanine, fluoro-amino acids, designer amino acidssuch as β-methyl amino acids, C-methyl amino acids, N-methyl aminoacids, and amino acid analogues in general. Non-natural amino acids alsoinclude amino acids having derivatized side groups. Furthermore, any ofthe amino acids in the protein can be of the D (dextrorotary) form or L(levorotary) form. Allelic variants of a protein sequence of a wild typeor mutant AGPase small or large subunit polypeptide of the presentinvention are also encompassed within the scope of the invention.

Amino acids can be generally categorized in the following classes:non-polar, uncharged polar, basic, and acidic. Conservativesubstitutions whereby a mutant AGPase small subunit polypeptide of thepresent invention and/or a wild type or mutant AGPase large subunitpolypeptide having an amino acid of one class is replaced with anotheramino acid of the same class fall within the scope of the subjectinvention so long as the polypeptide having the substitution stillretains substantially the same functional activity (e.g., enzymaticand/or increased heat stability of an AGPase enzyme) as the polypeptidethat does not have the substitution. Polynucleotides encoding a mutantAGPase small subunit polypeptide and/or a wild type or mutant AGPaselarge subunit polypeptide having one or more amino acid substitutions inthe sequence are contemplated within the scope of the present invention.Table 1 below provides a listing of examples of amino acids belonging toeach class.

TABLE 1 Class of Amino Acid Examples of Amino Acids Nonpolar Ala, Val,Leu, Ile, Pro, Met, Phe, Trp Uncharged Polar Gly, Ser, Thr, Cys, Tyr,Asn, Gln Acidic Asp, Glu Basic Lys, Arg, His

The subject invention also concerns variants of the polynucleotides ofthe present invention that encode functional wild type or mutant AGPasesmall or large subunit polypeptides of the invention. Variant sequencesinclude those sequences wherein one or more nucleotides of the sequencehave been substituted, deleted, and/or inserted. The nucleotides thatcan be substituted for natural nucleotides of DNA have a base moietythat can include, but is not limited to, inosine, 5-fluorouracil,5-bromouracil, hypoxanthine, 1-methylguanine, 5-methylcytosine, andtritylated bases. The sugar moiety of the nucleotide in a sequence canalso be modified and includes, but is not limited to, arabinose,xylulose, and hexose. In addition, the adenine, cytosine, guanine,thymine, and uracil bases of the nucleotides can be modified withacetyl, methyl, and/or thio groups. Sequences containing nucleotidesubstitutions, deletions, and/or insertions can be prepared and testedusing standard techniques known in the art.

Fragments and variants of a mutant polypeptide of the present inventioncan be generated as described herein and tested for the presence ofenzymatic and heat stable function using standard techniques known inthe art. Thus, an ordinarily skilled artisan can readily prepare andtest fragments and variants of a mutant polypeptide of the invention anddetermine whether the fragment or variant retains functional activityrelative to full-length or a non-variant mutant polypeptide.

Polynucleotides and polypeptides contemplated within the scope of thesubject invention can also be defined in terms of more particularidentity and/or similarity ranges with those sequences of the inventionspecifically exemplified herein. The sequence identity will typically begreater than 60%, preferably greater than 75%, more preferably greaterthan 80%, even more preferably greater than 90%, and can be greater than95%. The identity and/or similarity of a sequence can be 49, 50, 51, 52,53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70,71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% as compared to a sequenceexemplified herein. Unless otherwise specified, as used herein percentsequence identity and/or similarity of two sequences can be determinedusing the algorithm of Karlin and Altschul (1990), modified as in Karlinand Altschul (1993). Such an algorithm is incorporated into the NBLASTand XBLAST programs of Altschul et al. (1990). BLAST searches can beperformed with the NBLAST program, score=100, wordlength=12, to obtainsequences with the desired percent sequence identity. To obtain gappedalignments for comparison purposes, Gapped BLAST can be used asdescribed in Altschul et al. (1997). When utilizing BLAST and GappedBLAST programs, the default parameters of the respective programs(NBLAST and XBLAST) can be used. See NCBI/NIH website.

The subject invention also contemplates those polynucleotide moleculeshaving sequences which are sufficiently homologous with thepolynucleotide sequences exemplified herein so as to permithybridization with that sequence under standard stringent conditions andstandard methods (Maniatis et al., 1982). As used herein, “stringent”conditions for hybridization refers to conditions wherein hybridizationis typically carried out overnight at 20-25 C below the meltingtemperature (Tm) of the DNA hybrid in 6× SSPE, 5× Denhardt's solution,0.1% SDS, 0.1 mg/ml denatured DNA. The melting temperature, Tm, isdescribed by the following formula (Beltz et al., 1983):

Tm=81.5 C+16.6 Log[Na+]+0.41(% G+C)−0.61(% formamide)−600/length ofduplex in base pairs.

Washes are typically carried out as follows:

(1) Twice at room temperature for 15 minutes in 1× SSPE, 0.1% SDS (lowstringency wash).

(2) Once at Tm-20 C for 15 minutes in 0.2× SSPE, 0.1% SDS (moderatestringency wash).

As used herein, the terms “nucleic acid” and “polynucleotide” refer to adeoxyribonucleotide, ribonucleotide, or a mixed deoxyribonucleotide andribonucleotide polymer in either single- or double-stranded form, andunless otherwise limited, would encompass known analogs of naturalnucleotides that can function in a similar manner as naturally-occurringnucleotides. The polynucleotide sequences include the DNA strandsequence that is transcribed into RNA and the strand sequence that iscomplementary to the DNA strand that is transcribed. The polynucleotidesequences also include both full-length sequences as well as shortersequences derived from the full-length sequences. Allelic variations ofthe exemplified sequences also fall within the scope of the subjectinvention. The polynucleotide sequence includes both the sense andantisense strands either as individual strands or in the duplex.

Techniques for transforming plant cells with a gene are known in the artand include, for example, Agrobacterium infection, biolistic methods,electroporation, calcium chloride treatment, PEG-mediatedtransformation, etc. U.S. Pat. No. 5,661,017 teaches methods andmaterials for transforming an algal cell with a heterologouspolynucleotide. Transformed cells can be selected, redifferentiated, andgrown into plants that contain and express a polynucleotide of theinvention using standard methods known in the art. The seeds and otherplant tissue and progeny of any transformed or transgenic plant cells orplants of the invention are also included within the scope of thepresent invention.

The subject invention also concerns methods for producing a plant thatexhibits increased heat stability relative to a wild type plant, whereina polynucleotide encoding a mutant AGPase small subunit polypeptide ofthe present invention is introduced into a plant cell and thepolypeptide(s) encoded by the polynucleotide(s) is expressed. In oneembodiment, the plant cell comprises non-mutant genes encoding wild typeAGPase large subunit polypeptide. In another embodiment, the plant cellcomprises at least one polynucleotide encoding a mutant AGPase largesubunit polypeptide. In a further embodiment, a polynucleotide encodinga mutant AGPase large subunit polypeptide is also introduced into aplant cell along with the polynucleotide encoding the mutant AGPasesmall subunit polypeptide. In one embodiment, the polynucleotide orpolynucleotides is incorporated into the genome of the plant cell and aplant is grown from the plant cell. In a preferred embodiment, the plantgrown from the plant cell stably expresses the incorporatedpolynucleotide or polynucleotides.

The subject invention also concerns oligonucleotide probes and primers,such as polymerase chain reaction (PCR) primers, that can hybridize to acoding or non-coding sequence of a polynucleotide of the presentinvention. Oligonucleotide probes of the invention can be used inmethods for detecting and quantitating nucleic acid sequences encoding amutant AGPase small subunit polypeptide of the invention.Oligonucleotide primers of the invention can be used in PCR methods andother methods involving nucleic acid amplification. In a preferredembodiment, a probe or primer of the invention can hybridize to apolynucleotide of the invention under stringent conditions. Probes andprimers of the invention can optionally comprise a detectable label orreporter molecule, such as fluorescent molecules, enzymes, radioactivemoiety (e.g., ³H, ³⁵S, ¹²⁵I, etc.), and the like. Probes and primers ofthe invention can be of any suitable length for the method or assay inwhich they are being employed. Typically, probes and primers of theinvention will be 10 to 500 or more nucleotides in length. Probes andprimers that are 10 to 20, 21 to 30, 31 to 40, 41 to 50, 51 to 60, 61 to70, 71 to 80, 81 to 90, 91 to 100 or more nucleotides in length arecontemplated within the scope of the invention. Probes and primers ofthe invention can have complete (100%) nucleotide sequence identity withthe polynucleotide sequence, or the sequence identity can be less than100%. For example, sequence identity between a probe or primer and asequence can be 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80%, 75%, 70% or anyother percentage sequence identity so long as the probe or primer canhybridize under stringent conditions to a nucleotide sequence of apolynucleotide of the invention. In one embodiment, a probe or primer ofthe invention has 70% or greater, 75% or greater, 80% or greater, 85% orgreater, 90% or greater, or 95% to 100% sequence identity with anucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:11, SEQ IDNO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, or SEQ ID NO:16, or thecomplement thereof.

The subject invention also concerns isolated mutant AGPase small subunitpolypeptides. In one embodiment, the mutant AGPase small subunitpolypeptide is an AGPase small subunit polypeptide of Zea mays. In aspecific embodiment, an AGPase small subunit polypeptide of theinvention has an amino acid sequence as shown in SEQ ID NO:2, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9,SEQ ID NO:10, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, orSEQ ID NO:25, or functional fragment or variant thereof. An AGPase smallsubunit polypeptide or enzyme of the invention can be purified usingstandard techniques known in the art. In one embodiment, apolynucleotide of the invention encoding an AGPase small subunitpolypeptide is incorporated into a microorganism, such as E. coli, andthe AGPase small subunit polypeptide expressed in the microorganism andthen isolated therefrom.

In certain embodiments, polypeptides of the invention, and functionalpeptide fragments thereof, can be used to generate antibodies that bindspecifically to a polypeptide of the invention, and such antibodies arecontemplated within the scope of the invention. The antibodies of theinvention can be polyclonal or monoclonal and can be produced andisolated using standard methods known in the art.

Polypeptide fragments according to the subject invention typicallycomprise a contiguous span of about or at least 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84,85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101,102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115,116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129,130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143,144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157,158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171,172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185,186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199,200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213,214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227,228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241,242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255,256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269,270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283,284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297,298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311,312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325,326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339,340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353,354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367,368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381,382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395,396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409,410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423,424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437,438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451,452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465,466, 467, 468, 469, 470, 471, 472, 473, 474, or 475 amino acids of SEQID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ IDNO:8, SEQ ID NO:9, SEQ ID NO:10.

In certain embodiments, polypeptide fragments of the subject inventioncan be any integer in length from at least about 25 consecutive aminoacids to 1 amino acid less than the full-length sequence, such as thoseshown in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ IDNO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:10. Thus, for SEQ ID NO:2,SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ IDNO:9, or SEQ ID NO:10, a polypeptide fragment can be any integer ofconsecutive amino acids from about 25 to 475 amino acids. The term“integer” is used herein in its mathematical sense and thusrepresentative integers include: 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70,71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104,105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118,119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132,133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146,147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160,161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174,175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188,189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202,203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216,217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230,231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244,245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258,259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272,273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286,287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300,301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314,315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328,329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342,343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356,357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370,371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384,385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398,399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412,413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426,427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440,441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454,455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468,469, 470, 471, 472, 473, 474, and/or 475.

Each polypeptide fragment of the subject invention can also be describedin terms of its N-terminal and C-terminal positions. For example,combinations of N-terminal to C-terminal fragments of about 25contiguous amino acids to 1 amino acid less than the full lengthpolypeptide, such as those of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:10 areincluded in the present invention. Thus, using SEQ ID NO:2, SEQ ID NO:4,SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQID NO:10 as an example, a 25 consecutive amino acid fragment couldcorrespond to amino acids of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:10 selectedfrom the group consisting of 1-25, 2-26, 3-27, 4-28, 5-29, 6-30, 7-31,8-32, 9-33, 10-34, 11-35, 12-36, 13-37, 14-38, 15-39, 16-40, 17-41,18-42, 19-43, 20-44, 21-45, 22-46, 23-47, 24-48, 25-49, 26-50, 27-51,28-52, 29-53, 30-54, 31-55, 32-56, 33-57, 34-58, 35-59, 36-60, 37-61,38-62, 39-63, 40-64, 41-65, 42-66, 43-67, 44-68, 45-69, 46-70, 47-71,48-72, 49-73, 50-74, 51-75, 52-76, 53-77, 54-78, 55-79, 56-80, 57-81,58-82, 59-83, 60-84, 61-85, 62-86, 63-87, 64-88, 65-89, 66-90, 67-91,68-92, 69-93, 70-94, 71-95, 72-96, 73-97, 74-98, 75-99, 76-100, 77-101,78-102, 79-103, 80-104, 81-105, 82-106, 83-107, 84-108, 85-109, 86-110,87-111, 88,-112, 89-113, 90-114, 91-115, 92-116, 93-117, 94-118, 95-119,96-120, 97-121, 98-122, 99-123, 100-124, 101-125, 102-126, 103-127,104-128, 105-129, 106-130, 107-131, 108-132, 109-133, 110-134, 111-135,112-136, 113-137, 114-138, 115-139, 116-140, 117-141, 118-142, 119-143,120-144, 121-145, 122-146, 123-147, 124-148, 125-149, 126-150, 127-151,128-152, 129-153, 130-154, 131-155, 132-156, 133-157, 134-158, 135-159,136-160, 137-161, 138-162, 139-163, 140-164, 141-165, 142-166, 143-167,144-168, 145-169, 146-170, 147-171, 148-172, 149-173, 150-174, 151-175,152-176, 153-177, 154-178, 155-179, 156-180, 157-181, 158-182, 159-183,160-184, 161-185, 162-186, 163-187, 164-188, 165-189, 166-190, 167-191,168-192, 169-193, 170-194, 171-195, 172-196,. 173-197, 174-198, 175-199,176-200, 177-201, 178-202, 179-203, 180-204, 181-205, 182-206, 183-207,184-208, 185-209, 186-210, 187-211, 188-212, 189-213, 190-214, 191-215,192-216, 193-217, 194-218, 195-219, 196-220, 197-221, 198-222, 199-223,200-224, 201-225, 202-226, 203-227, 204-228, 205-229, 206-230, 207-231,208-232, 209-233, 210-234, 211-235, 212-236, 213-237, 214-238, 215-239,216-240, 217-241, 218-242, 219-243, 220-244, 221-245, 222-246, 223-247,224-248, 225-249, 226-250, 227-251, 228-252, 229-253, 230-254, 231-255,232-256, 233-257, 234-258, 235-259, 236-260, 237-261, 238-262, 239-263,240-264, 241-265, 242-266, 243-267, 244-268, 245-269, 246-270, 247-271,248-272, 249-273, 250-274, 251-275, 252-276, 253-277, 254-278, 255-279,256-280, 257-281, 258-282, 259-283, 260-284, 261-285, 262-286, 263-287,264-288, 265-289, 266-290, 267-291, 268-292, 269-293, 270-294, 271-295,272-296, 273-297, 274-298, 275-299, 276-300, 277-301, 278-302, 279-303,280-304, 281-305, 282-306, 283-307, 284-308, 285-309, 286-310, 287-311,288-312, 289-313, 290-314, 291-315, 292-316, 293-317, 294-318, 295-319,296-320, 297-321, 298-322, 299-323, 300-324, 301-325, 302-326, 303-327,304-328, 305-329, 306-330, 307-331, 308-332, 309-333, 310-334, 311-335,312-336, 313-337, 314-338, 315-339, 316-340, 317-341, 318-342, 319-343,320-344, 321-345, 322-346, 323-347, 324-348, 325-349, 326-350, 327-351,328-352, 329-353, 330-354, 331-355, 332-356, 333-357, 334-358, 335-359,336-360, 337-361, 338-362, 339-363, 340-364, 341-365, 342-366, 343-367,344-368, 345-369, 346-370, 347-371, 348-372, 349-373, 350-374, 351-375,352-376, 353-377, 354-378, 355-379, 356-380, 357-381, 358-382, 359-383,360-384, 361-385, 362-386, 363-387, 364-388, 365-389, 366-390, 367-391,368-392, 369-393, 370-394, 371-395, 372-396, 373-397, 374-398, 375-399,376-400, 377-401, 378-402, 379-403, 380-404, 381-405, 382-406, 383-407,384-408, 385-409, 386-410, 387-411, 388-412, 389-413, 390-414, 391-415,392-416, 393-417, 394-418, 395-419, 396-420, 397-421, 398-422, 399-423,400-424, 401-425, 402-426, 403-427, 404-428, 405-429, 406-430, 407-431,408-432, 409-433, 410-434, 411-435, 412-436, 413-437, 414-438, 415-439,416-440, 417-441, 418-442, 419-443, 420-444, 421-445, 422-446, 423-447,424-448, 425-449, 426-450, 427-451, 428-452, 429-453, 430-454, 431-455,432-456, 433-457, 434-458, 435-459, 436-460, 437-461, 438-462, 439-463,440-464, 441-465, 442-466, 443-467, 444-468, 445-469, 446-470, 447-471,448-472, 449-473, 450-474, and/or 451-475. Similarly, the amino acidscorresponding to all other fragments of sizes between 26 consecutiveamino acids and 474 (or 475) consecutive amino acids of SEQ ID NO:2, SEQID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ IDNO:9, or SEQ ID NO:10 are included in the present invention and can alsobe immediately envisaged based on these examples. Therefore, additionalexamples, illustrating various fragments of the polypeptides of SEQ IDNO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8,SEQ ID NO:9, or SEQ ID NO:10 are not individually listed herein in orderto avoid unnecessarily lengthening the specification.

Polypeptide fragments comprising: 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69,70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87,88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104,105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118,119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132,133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146,147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160,161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174,175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188,189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202,203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216,217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230,231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244,245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258,259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272,273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286,287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300,301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314,315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328,329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342,343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356,357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370,371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384,385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398,399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412,413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426,427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440,441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454,455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468,469, 470, 471, 472, 473, and 474 (or 475) consecutive amino acids of SEQID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ IDNO:8, SEQ ID NO:9, or SEQ ID NO:10 may alternatively be described by theformula “n to c” (inclusive), where “n” equals the N-terminal amino acidposition and “c” equals the C-terminal amino acid position of thepolypeptide. In this embodiment of the invention, “n” is an integerhaving a lower limit of 1 and an upper limit of the total number ofamino acids of the full length polypeptide minus 24 (e.g., 475-24=451for SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:8; 476-24=452for SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, or SEQ ID NO:10). “c” is aninteger between 25 and the number of amino acids of the full lengthpolypeptide sequence (475 for SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, orSEQ ID NO:8; 476 for SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, or SEQ IDNO:10) and “n” is an integer smaller than “c” by at least 24. Therefore,for SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQID NO:8, SEQ ID NO:9, or SEQ ID NO:10, “n” is any integer selected fromthe list consisting of: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68,69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86,87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103,104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117,118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131,132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145,146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159,160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173,174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187,188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201,202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215,216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229,230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243,244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257,258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271,272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285,286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299,300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313,314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327,328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341,342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355,356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369,370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383,384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397,398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411,412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425,426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439,440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, and 451 (or 452);and “c” is any integer selected from the group consisting of: 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62,63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80,81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98,99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112,113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126,127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140,141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154,155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168,169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182,183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196,197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210,211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224,225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238,239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252,253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266,267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280,281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294,295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308,309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322,323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336,337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350,351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364,365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378,379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392,393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406,407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420,421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434,435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448,449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462,463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, and 475 (or 476)provided that “n” is a value less than “c” by at least 24. Everycombination of “n” and “c” positions are included as specificembodiments of polypeptide fragments of the invention. All ranges usedto describe any polypeptide fragment embodiment of the present inventionare inclusive unless specifically set forth otherwise.

Fragments of a mutant AGPase small subunit polypeptide of the inventionor an AGPase large subunit polypeptide, as described herein, can beobtained by cleaving the polypeptides of the invention with aproteolytic enzyme (such as trypsin, chymotrypsin, or collagenase) orwith a chemical reagent, such as cyanogen bromide (CNBr). Alternatively,polypeptide fragments can be generated in a highly acidic environment,for example at pH 2.5. Polypeptide fragments can also be prepared bychemical synthesis or using host cells transformed with an expressionvector comprising a polynucleotide encoding a fragment of an AGPaselarge subunit polypeptide or a fragment of a mutant AGPase small subunitpolypeptide of the invention, for example, a mutant polypeptide that isa fragment of the amino acid sequence shown in SEQ ID NO:2, SEQ ID NO:4,SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ IDNO:10, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, or SEQ IDNO:25. Fragments of a mutant large or small subunit AGPase polypeptideof the invention also contemplated herein include fragments of thepolypeptide wherein all or a part of a transit or signal sequence of thepolypeptide is removed.

The subject invention also concerns cells transformed with apolynucleotide of the present invention encoding a mutant AGPase smallsubunit polypeptide of the invention. In one embodiment, the cell istransformed with a polynucleotide sequence comprising a sequenceencoding the amino acid sequence shown in SEQ ID NO:2, SEQ ID NO:4, SEQID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ IDNO:10, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ′ID NO:24, or SEQ IDNO:25, or a functional fragment or variant thereof. In a specificembodiment, the cell is transformed with a polynucleotide sequence shownin SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13,SEQ ID NO:14, SEQ ID NO:15, or SEQ ID NO:16, or a sequence encoding afunctional fragment or variant of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5,SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ IDNO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, or SEQ ID NO:25. In oneembodiment, a cell is also transformed with a polynucleotide encoding amutant AGPase large subunit polypeptide as described herein. In oneembodiment, the polynucleotide sequence is provided in an expressionconstruct of the invention. The transformed cell can be a prokaryoticcell, for example, a bacterial cell such as E. coli or B. subtilis, orthe transformed cell can be a eukaryotic cell, for example, a plantcell, including protoplasts, or an animal cell. Plant cells include, butare not limited to, dicotyledonous, monocotyledonous, and conifer cells.In one embodiment, the plant cell is a cell from a Zea mays plant.Animal cells include human cells, mammalian cells, avian cells, andinsect cells. Mammalian cells include, but are not limited to, COS, 3T3,and CHO cells.

The subject invention also concerns methods for increasing starchsynthesis in a plant or plant tissue (such as a plant seed or endospermtissue). In one embodiment, a method of the invention comprisesintroducing one or more polynucleotides of the present invention into aplant. In certain embodiments, the polynucleotides introduced into theplant encode one or more polypeptides comprising the amino acid sequenceshown in any of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:21, SEQ IDNO:22, SEQ ID NO:23, SEQ ID NO:24, or SEQ ID NO:25, or a fragment orvariant thereof In a specific embodiment, the polynucleotide comprisesthe nucleotide sequence shown in SEQ ID NO:1, or SEQ ID NO:3, or afragment or variant thereof In further specific embodiments, thepolynucleotide comprises the nucleotide sequences shown in SEQ ID NO:11,SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, or SEQ ID NO:16,or a fragment or variant thereof. In one embodiment, the polynucleotideis stably incorporated into the genome of the plant or plant tissue. Thepolynucleotide can comprise regulatory elements, such as a promoterand/or enhancer sequences, that provide for increased expression of thepolynucleotide and/or the polypeptide encoded thereby. In a specificembodiment, the promoter sequence is one that provides for constitutiveor tissue-specific (e.g., endosperm) expression. Plants or plant tissuescontaining the polynucleotide, or progeny of the plants, optionally canbe screened for increased expression of a polynucleotide or polypeptideof the invention. In one embodiment, multiple copies of one or morepolynucleotides of the invention are introduced into a plant or planttissue and stably incorporated into the genome of the plant. In oneembodiment, a polynucleotide of the invention is provided in anexpression construct as described herein.

Single letter amino acid abbreviations are defined in Table 2.

TABLE 2 Letter Symbol Amino Acid A Alanine B Asparagine or aspartic acidC Cysteine D Aspartic Acid E Glutamic Acid F Phenylalanine G Glycine HHistidine I Isoleucine K Lysine L Leucine M Methionine N Asparagine PProline Q Glutamine R Arginine S Serine T Threonine V Valine WTryptophan Y Tyrosine Z Glutamine or glutamic acid

Materials and Methods Random Mutagenesis

Mutations were introduced into Sh2 and Bt2 by PCR random mutagenesis(GeneMorph II EZClone Domain Mutagenesis Kit, Stratagene). A mixture ofnon-biased, error-prone DNA polymerases was used to introduce pointmutations. Wildtype Sh2 and Bt2 coding sequences in pMONcSh2 andpMONcBt2 (Giroux et al., 1996) respectively were used as templates forPCR. Two pairs of primers (Sh2: 5′-GAAGGAGATATATCCATGG-3′ (SEQ IDNO:17), 5′-GGATCCCCGGGTACCGAGCTC-3′ (SEQ ID NO:18) Bt2:5′-GAAGGAGATATATCCATGG-3′ (SEQ ID NO:19), 5′-GTTGATATCTGAATTCGAGCTC-3′(SEQ ID NO:20)) flanking Sh2 and Bt2 were used for error-prone PCR.Mutant Sh2 clones produced by PCR were subcloned into vector pMONcSh2according to Stratagene protocols. pMONcSh2 was then used to transformE. coli strain AC70R1-504 that contained wildtype Bt2 in the compatiblevector pMONcBt2. Mutant Bt2 clones produced by PCR were subcloned intovector pMONcBt2. pMONcBt2 was then used to transform E. coli strainAC70R1-504 that contained wildtype Sh2 in the compatible vectorpMONcSh2.

Bacterial Expression System

A bacterial expression system (Iglesias et al., 1993) allowed us torandomly mutagenize maize endosperm AGPase genes and score AGPaseactivity in a fast and efficient way by exposing plates to iodine vaporsas described below. The E. coli system is ideal for studying plantAGPases for a number of reasons discussed in Georgelis et al. (2007).

Glycogen Detection

Glycogen synthesis was detected by production of brown staining coloniesfollowing exposure to iodine vapors. E. coli cells were grown onKornberg media in the presence of 75 μg/mL spectinomycin, 50 μg/mLkanamycin and 1% w/v glucose for 16 h at 37° C. (Govons et al., 1969).The colonies were exposed to iodine vapors for 1 min. Colonies withinactive AGPase produced no color following exposure to iodine vaporswhile active AGPase produced glycogen and, in turn, brown staining withiodine. AGPase variants staining darker than wildtype were selected forfurther study.

Glycogen Quantitation

Glycogen quantitation was performed by phenol reaction (Hanson andPhillips, 1981). In brief, glycogen was extracted from 1.6 ml of E. colicells (OD₆₀₀=2.0) grown in LB containing 2% w/v glucose by boiling for 3hours in 50% w/v KOH. Glycogen was then precipitated by adding ethanolto 70% v/v and centrifuging at 10000×g for 10 min. After pellet drying,200 μl de-ionized water, 200 μl of 5% w/v phenol and 1 mL ofconcentrated sulfuric acid were added. Glycogen was estimated by theabsorbance at 488 nm.

DNA Sequencing

Sh2 and Bt2 mutants that produced enhanced glycogen were double-passsequenced by the Genome Sequencing Services Laboratory (GSSL) of theInterdisciplinary Center for Biotechnology Research at the University ofFlorida. Data analysis was performed by Bioedit software (Hall, 1999).

Purification of Maize Endosperm AGPase from AC70R1-504 E. coli Cells

AC70R1-504 E. coli cells expressing maize endosperm AGPase were grown in2 L of Luria-Broth (LB) medium in the presence of 75 μg/mLspectinomycin, 50 μg/mL kanamycin and 2% w/v glucose for 16 h at 37° C.with shaking. At OD600=0.6, 0.2 mM isopropyl-beta-D-thiogalactoside(IPTG) and 0.02 mg/mL nalidixic acid were added to induce proteinexpression. The cultures were immediately moved to room temperature andgrown for 4 h with shaking. The following steps were conducted at 4° C.Cells were harvested by centrifuging at 3000×g and the pellet wasresuspended in 16 mL of buffer A (50 mM KH₂PO₄ pH 7.0, 5 mM MgCl₂, 0.5mM EDTA) and protease inhibitors (1 μg/mL pepstatin, 0.1 mM PMSF, 10μg/mL chymostatin, and 1 mM benzamidine). The cells were lysed with aFrench press and centrifuged at 26000×g. The protein concentration ofthe supernatant was adjusted to 30 mg/mL by adding buffer A. Threetenths of volume of 1% protamine sulfate were added and the mixturestirred on ice for 20 min and then centrifuged at 26000×g for 20 min.The supernatant was brought to 45% saturation with ammonium sulfate,stirred on ice for 20 min and centrifuged at 26000×g for 20 min. Thepellet was re-suspended in 2-2.5 mL of buffer A. The mixture was passedthrough a strong anion exchange column (macro-prep High Q support,Biorad), and an Econo-pac hydroxyapatite cartridge (Biorad) as describedby Boehlein et al. (2005). AGPase was desalted by using Zeba MicroDesalt Spin Columns (Pierce) before assaying. AGPase was exchanged into50 mM HEPES, 5 mM MgCl₂, 0.5 mM EDTA and 0.5 mg/mL BSA (for stability).

Kinetic Characterization of AGPase

The forward direction of the reaction was used(G-1-P+ATP→ADP-glucose+PPi) for estimating K_(cat), K_(m) for ATP andG-1-P, and affinities for 3-PGA and Pi. More specifically, 0.04-0.06 μgof purified AGPase was assayed for specific activity in a total volumeof 200 μl of 50 mM HEPES pH 7.4, 15 mM MgCl₂, 1.0 mM ATP, and 2.0 mMG-1-P at 37° C. for 10 min. For determining K_(m)s for ATP and G-1-P andK_(a) for 3-PGA, varying amounts of ATP, G-1-P and 3-PGA respectively.K_(i) for Pi was estimated by adding various amounts of Pi, 1 mM ATP, 2mM G-1-P, and 2.5 mM 3-PGA were used. The reaction was stopped byboiling for 2 min. PPi was estimated by coupling the reaction to areduction in NADH concentration using 300 μl of coupling reagent. Thecoupling reagent contained 25 mM imidazole pH 7.4, 4 mM MgCl₂, 1 mMEDTA, 0.2 mM NADH, 0.725U aldolase, 0.4U triose phosphate isomerase,0.6U glycerophosphate dehydrogenase, 1 mM fructose 6-phosphate and 0.8μg of pyrophosphate dependent phosphofructokinase (PPi-PFK). All theenzymes were purchased from Sigma except for PPi-PFK which was producedand purified according to Deng et al. 1999 with some modifications(Boehlein and Hannah, unpublished data). NADH concentration wasestimated by absorbance at 340 nm. PPi concentration was calculated by astandard curve developed by using various amounts of PPi instead ofAGPase. The amount of PPi produced by AGPase was linear with time andenzyme concentration. The kinetic constants of AGPase were calculated byPrism 4.0 (Graph Pad, San Diego Calif.).

Measuring AGPase Specific Activity from Crude or Partially PurifiedProtein Extracts

AC70R1-504 E. coli cells expressing maize endosperm AGPase were grown in2 L of Luria-Broth (LB) medium in the presence of 75 μg/mLspectinomycin, 50 μg/mL kanamycin and 2% w/v glucose at 37° C. withshaking until OD600=2.0. Gene expression was not induced. The followingsteps were conducted at 4° C. Cells were harvested by centrifuging at3000×g and the pellet was resuspended in 16 mL of buffer A (50 mM KH₂PO₄pH 7.0, 5 mM MgCl₂, 0.5 mM EDTA) and protease inhibitors (1 μg/mLpepstatin, 0.1 mM PMSF, 10 μg/mL chymostatin, and 1 mM benzamidine). Thecells were lysed with a French press and centrifuged at 26000×g. AGPaseactivity of the crude extract was measured from the supernatant storedat −80° C. The rest of the supernatant was partially purified throughprotamine sulfate and ammonium sulfate as described above. Proteinextracts were desalted as described by Boehlein et al. (2005) beforeassay and were exchanged into 50 mM HEPES, 5 mM MgCl₂, and 0.5 mM EDTA.AGPase specific activity was monitored in the reverse direction(ADP-glucose+PPi→G-1-P+ATP) using saturating amounts of substrates andactivator as described by Boehlein et al. (2005).

Determining Heat Stability of AGPase

AGPase was purified as described above. AGPase was further diluted 1/100(v/v) in 50 mM HEPES, 5 mM MgCl₂, 0.5 mM EDTA and 0.5 mg/mL BSA and heattreated at 42 or 53° C. for various times, and then cooled on ice. Theactivity remaining after heat treatment was monitored in the forward andreverse direction by using saturating amounts of ATP, G-1-P and 3-PGA.The data were plotted as log of percentage of remaining activity versustime of heat treatment. The inactivation constant t₁₁₂ was calculatedfrom the formula t_(1/2)=0.693/(−2.3*slope).

Qualitative Determination of the AGPase Purity

The purity of AGPase was monitored in the following way. Six μg ofAGPase were diluted 1:1 in denaturing solution (100 mM Tris-Cl pH 6.8,4% SDS, and 8 mM DTT), heated at 95° C. for 5 min, electrophoresed on a5% SDS polyacrylamide gel at 150V for 1 h and visualized by stainingwith Coomassie Brilliant Blue (Laemmli, 1970).

Protein Blot Analysis of Crude Extracts

Samples were vacuum blotted onto a PVDF membrane (Biorad) by usingHybri-Dot blot apparatus (Life Technologies). The PVDF membrane had beenpre-soaked in methanol for 5 min and then in transfer buffer [20% (v/v)methanol, 0.303% (w/v) Tris and 1.44% (w/v) glycine] for 10 min. Themembrane was incubated with blocking buffer [0.8% (w/v) NaCl, 0.02%(w/v) KCl, 0.144% (w/v) Na₂HPO₄, 0.024% (w/v) KH₂PO₄, 5% (w/v) bovineserum albumin (BSA), and 0.05% (v/v) Tween-20] for 1 h with constantshaking. The blot was incubated with blocking buffer containing 1:10000(v/v) of monoclonal antibody against BT2 (kindly provided by SueBoehlein) for lh with shaking. Then, the blot was washed 3×10 min withwashing buffer (blocking buffer—BSA) with constant shaking. The blot wasthen incubated with a 1:60000 dilution of goat anti-mouse secondaryantibody conjugated with horseradish peroxidase (Pierce) for 45 min.Finally the blot was washed 3×10 min. Proteins were visualized using anenhanced chemiluminescent substrate kit (Pierce).

3D Modeling

BT2 monomer structure was modeled after the potato small unit in therecently-published three dimensional structure of the potato tuberhomotetrameric AGPase (RCSB Protein Data Bank #:1YP2). Homology modelingwas done by using SWISS MODEL (Peitsch, 1995; Guex and Peitsch, 1997;Schwede et al. 2003; Kopp and Schwede, 2004; Arnold et al, 2006). Aminoacid 462 (Thr or Ile) contacting residues were determined by using Jmol,an open-source Java viewer for chemical structures in 3D,(http://www.jmol.org/).

Yeast Two Hybrid

Yeast transformations and a β-galactosidase assay were conducted asdescribed by Greene and Hannah (1998b). The only modification was theuse of pGBKT7 and pGADT7 as vectors for the bait and the preyrespectively. pGBKT7-53 and pGADT7-T plasmids were used as a positivecontrol.

All patents, patent applications, provisional applications, andpublications referred to or cited herein are incorporated by referencein their entirety, including all figures and tables, to the extent theyare not inconsistent with the explicit teachings of this specification.

Following are examples that illustrate procedures for practicing theinvention. These examples should not be construed as limiting. Allpercentages are by weight and all solvent mixture proportions are byvolume unless otherwise noted.

EXAMPLE 1

A mutant Bt2 library was created by error-prone PCR. The mutational loadwas ˜2 non-synonymous mutations per clone (Georgelis et al. 2007). Themutants were expressed in E. coli along with a wildtype Sh2 gene.Approximately 50,000 colonies were screened for glycogen production. Tendark staining colonies were picked. The two darkest staining Bt2 mutantswere sequenced. Both had the same non-synonymous mutation resulting in achange of amino acid 462 from threonine to isoleucine (TI). Thethreonine in that position is absolutely conserved among the higherplant small subunits (data not shown). BT2-TI/SH2 (BT2 comprising the TImutation and complexed with SH2) produced more glycogen than did BT2/SH2(FIG. 1). Cells expressing BT2-TI and BT2, as homotetramers, did notproduce detectable amounts of glycogen (FIG. 1). This indicates that theamount of E. coli-synthesized glycogen depends exclusively on thecomplex of BT2-TI or BT2 with SH2.

A dot-blot of the crude extracts from cells expressing BT2/SH2 andBT2-TI/SH2 indicated that BT2-TI is found in higher amounts in E. coli(FIGS. 2A-2B). While AGPase activity levels of crude extracts fromnon-induced cells expressing BT2/SH2 and BT2-TI/SH2 were too low todetect, the partially purified extract from BT2-TI/SH2 had 20 times moreactivity than did the partially purified extract from BT2/SH2 (FIG. 3).The possibility that BT2-TI/SH2 produced more protein and activitybecause of more efficient transcription/translaton is unlikely since thecodons ACA (T) to ATA (I) are used with the same frequency in E. coli(6.1 and 5.0% respectively) (Nakamura et al., 2000). This suggests thatthe higher amount of protein and activity in BT2-TI/SH2 cells is due toincreased stability of the AGPase.

To determine the kinetic properties and heat stability of BT2-TI/SH2 anddecipher the cause of enhanced glycogen synthesis in E. coli,recombinant BT2-TI/SH2 and BT2/SH2 AGPases were purified (FIG. 4). Thekinetic properties of BT2-TI/SH2, as summarized in Table 3, show thatthe K_(m) for G-1-P and ATP, K_(a) for 3-PGA and K_(i) for Pi wereindistinguishable from BT2/SH2. Surprisingly, the k_(cat) of BT2-TI is30% lower than the k_(cat) of BT2/SH2. These kinetic properties thencannot account for the darker staining of BT2-TI/SH2 in E. coli.However, BT2-TI/SH2 is clearly more heat-stable than BT2/SH2 (FIGS.5A-5B). These results strongly suggest that the high heat stability ofBT2-TI/SH2 accounts for the enhanced amount of glycogen in E. coli.

MP is a small subunit variant that can lead to agronomic gain. Some ofits features include increased activity in the absence of the activator3-PGA, increased affinity for 3-PGA, decreased affinity for Pi (Table 3)and elevated heat stability compared to BT2/SH2 (FIG. 3) (Cross et al.,2004; Boehlein et al., 2005). Since BT2-TI/SH2 was not as heat-stable asMP/SH2 (FIG. 3), the amino acid change of TI was introduced into MP inan effort to further increase the heat stability of MP (MP-TI).

Cells expressing MP-TI/SH2 (MP having the TI mutation and complexed withSH2) produced the same amount of glycogen as cells expressing MP/SH2(FIG. 1). However, greater amounts of the MP-TI protein relative to BT2were found in crude extracts of E. coli expressing the two proteins withSH2 (FIGS. 2A-2B). The activity of the crude extracts and the partiallypurified extracts from MP-TI/SH2 was 2-3 fold higher than from MP/SH2(FIG. 3). MP-TI/SH2, in its pure form (FIG. 4), maintained the favorablekinetic properties of MP/SH2 (Table 3) except that its k_(cat) wasreduced ˜30% compared to MP/SH2. Additionally, MP-TI/SH2 exhibitsgreater heat stability than does MP/SH2 (FIGS. 6A-6B).

The crystal structure of maize endosperm AGPase has not been resolved.The only relevant structure is a potato tuber small subunit homotetramer(Jin et al., 2005). The potato tuber small subunit shows 88% identityand 96% similarity to BT2. BT2 monomer structure was modeled after theresolved structure of the potato tuber small subunit (FIG. 7A). Theresidue mutated in TI is part of a β-helix and it makes hydrophobiccontact with two residues (Pro, Leu) of the N-terminus of the smallsubunit (FIG. 7B). The amino acid change from Thr to Ile in TI shortensthe distance from the Pro and Leu mentioned above (FIG. 7C). It istempting to speculate that the TI mutation strengthens the hydrophobicinteraction between the C- and the N-terminus of the small subunit andresults in greater stability. Unlike MP, whose heat stability isattributed to residues at/near the subunit-subunit interfaces, TI maynot directly affect subunit-subunit interactions since it is far fromthe subunit-subunit interfaces (FIG. 7A).

To determine whether TI affects the strength of subunit-subunitinteractions SH2 was used as a bait and BT2, TI, and MP were used as aprey in a yeast two-hybrid system (Y2H). A quantitative β-galactosidaseassay indicated that, in contrast to MP, TI did not increase thestrength of subunit-subunit interactions (FIG. 8).

Finally, replacements of the original threonine by amino acids withshorter side chains such as serine, alanine and glycine did not affectthe heat stability of AGPase even though they did result in 10-fold ormore reduction in k_(cat) (data not shown). This indicates that theoriginal threonine in position 462 is important for AGPase activity butnot for heat stability.

EXAMPLE 2

The subject invention provides for agronomically important plant AGPasevariants by using random mutagenesis and a heterologous bacterialexpression system. BT2-TI was isolated as a small subunit variant thatincreased the amount of glycogen produced by E. coli cells whenexpressed along with SH2. Cells expressing BT2-TI/SH2 had 20-fold higherAGPase activity than cells expressing BT2/SH2. A dot-blot indicated thatthe crude protein extract from cells expressing BT2-TI/SH2 had moredetectable BT2 protein compared to cells expressing BT2/SH2. This resultcould be attributed to more efficient transcription/translation or togreater AGPase stability and/or solubility. As mentioned previously, amore efficient transcription/translation is unlikely based on codonusage. On the other hand, it was showed that the purified form ofBT2-TI/SH2 was significantly more heat-stable than the purified form ofBT2/SH2. This may render the BT2-TI/SH2 complex less prone toproteolysis and/or aggregation compared to BT2/SH2 in E. coli.

The kinetic and allosteric properties of BT2-TI/SH2 wereindistinguishable from BT2/SH2 except for a 30% lower k_(cat). The20-fold increase in AGPase activity of BT2-TI/SH2 expressing cells isinterpreted as a higher number of active AGPase molecules compared toBT2/SH2 expressing cells. This also means that less than 5% of thepotential AGPase molecules actually function in BT2/SH2.

It has been reported that the potato tuber small subunit can form ahomotetramer that has significant activity when given extremely highamounts of the activator 3-PGA (Ballicora et al., 1995). As shownherein, E. coli cells expressing BT2 and BT2-TI as homotetramers do notproduce detectable amounts of glycogen. Hence, the increased amounts ofglycogen observed in cells expressing BT2-TI/SH2 compared to cellsexpressing BT2/SH2 is due to the complex of BT2-TI with SH2 rather thanthe BT2-TI homotetramer.

Another small subunit variant that results in increased heat stabilityin a complex with SH2 is MP. The heat stability conferred by MP has beenmapped to residues near or at the subunit-subunit interaction interfaces(Boehlein, Shaw, Stewart, and Hannah, unpublished data). In contrast,the amino acid change of BT2-TI is far from these interfaces. Structuremodeling suggests that the TI change strengthens the intra-subunithydrophobic interactions between the C- and the N-terminus. The resultsfrom Y2H support the idea that, in contrast to MP, TI does notstrengthen the subunit-subunit interactions. However, the possibilitythat TI indirectly affects subunit-subunit interactions through aconformational change cannot be dismissed. It is possible that Y2H maynot be sensitive enough to reveal a difference in subunit-subunitinteractions between TI/SH2 and BT2/SH2 at the yeast growth temperatureof 30° C.

A comparison of the heat stability of pure BT2-TI/SH2 and MP/SH2indicated that BT2-TI/SH2 was not as heat-stable as MP/SH2.Additionally, MP/SH2 has several advantages not shared by BT2-TI/SH2,such as activity in the absence of the activator 3-PGA, a higheraffinity for 3-PGA, a lower affinity for the inhibitor Pi and a higherk_(cat) compared to BT2/SH2. It was investigated whether the heatstability of MP/SH2 could be further improved by introducing the TIchange into MP. The resulting variant, MP-TI, when expressed with SH2 inE. coli, yielded an equal amount of glycogen as MP/SH2. This could meanthat either MP-TI/SH2 was not more heat-stable than MP/SH2 or that theproduction of ADP-glucose catalyzed by AGPase was not limiting in E.coli anymore. The latter interpretation may be favored since AGPaseactivity in crude and partially purified extracts of cells expressingMP-TI/SH2 was 2-3 fold higher than cells expressing MP/SH2. MP-TI/SH2maintained all the kinetic and allosteric properties of MP/SH2 with theexception a 30% lower k_(cat). Most importantly, MP-TI/SH2 was moreheat-stable than MP/SH2. Two phases of heat stability in both MP-TI/SH2and MP/SH2 were observed. These phases were probably a result ofdifferent states of AGPase. This biphasic mode of heat stability hasbeen observed before and it is not specific to MP/SH2 or MP-TI (Boehleinet al. 2008). The first phase shows lower heat stability than does thesecond one. MP-TI/SH2 is more heat-stable in both phases compared toMP/SH2. However, what exactly the state of AGPase is in each phaseremains enigmatic. The biphasic mode of heat stability was not observedin BT2/SH2 and BT2-TI/SH2 because the samples were not heated for longenough time to reach the second phase and they were heated at lowertemperature than MP/SH2 and MP-TI/SH2 (42° C. instead of 53° C.).

Table 3 shows the kinetic properties of purified recombinant AGPasevariants. The kinetic and allosteric properties of AGPase variants weredetermined in the forward direction. k_(cat) (s⁻¹)(G-1-P) was estimatedby varying the amount of G-1-P and keeping ATP at saturating amounts (1mM). k_(cat) (s⁻¹) (ATP) was estimated by varying the amount of ATP andkeeping G-1-P at saturating amounts (2 mM). K_(i)'s are expressed asmean (95% confidence interval). All other values are expressed asmean±standard deviation. The specific activity of AGPase in the absenceof 3-PGA is expressed as a percentage of the specific activity in thepresence of 10 mM of 3-PGA (mean±standard error).

TABLE 3 BT2/SH2 BT2-TI/SH2 MP/SH2 MP-TI/SH2 K_(m) G-1-P (mM) 0.050 0.0400.079 0.059 (±0.008) (±0.006) (±0.007) (±0.005) k_(cat) (s⁻¹) (G-1-P)38.170 26.200 62.655 42.880 (±1.323) (±1.401) (±2.569) (±1.880) K_(m)ATP (mM) 0.102 0.146 0.133 0.112 (±0.020) (±0.050) (±0.021) (±0.013)k_(cat) (s⁻¹) (ATP) 43.321 29.112 69.337 49.031 (±1.554) (±1.030)(±3.276) (±1.903) K_(a) 3-PGA (mM) 0.480 0.330 0.100 0.068 (±0.137)(±0.060) (±0.010) (±0.010) K_(i) Pi (mM) 2.320 4.070 6.610 5.870 (0.530,4.100) (2.120, 6.020) (4.530, 8.690) (4.410, 7.330) V_(max) −3-PGA/V_(max) + 3-PGA (nmol/min/mg) BT2/SH2 BT2-TI/SH2 MP/SH2 MP-TI/SH2280/4000 134/2737 1856/6584 1101/4513 (7.2 ± 3.2%) (4.9 ± 1.5%) (28.2 ±3.1%) (24.4 ± 5.3%)

It should be understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication and the scope of the appended claims. In addition, anyelements or limitations of any invention or embodiment thereof disclosedherein can be combined with any and/or all other elements or limitations(individually or in any combination) or any other invention orembodiment thereof disclosed herein, and all such combinations arecontemplated with the scope of the invention without limitation thereto.

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1. A polynucleotide encoding a mutant plant AGPase small subunitprotein, or a functional fragment of said protein, said proteincomprising an amino acid mutation wherein the amino acid correspondingto the threonine amino acid at position 462 of wild type maize endospermAGPase small subunit protein is replaced by an amino acid that confersincreased heat stability when said mutant AGPase small subunit isexpressed to form an AGPase enzyme.
 2. The polynucleotide according toclaim 1, wherein said replacement amino acid that confers increased heatstability is an isoleucine.
 3. The polynucleotide according to claim 1,wherein said mutant AGPase small subunit is maize endosperm AGPase smallsubunit.
 4. The polynucleotide according to claim 1, wherein said mutantplant AGPase small subunit protein encoded by said polynucleotidecomprises the amino acid sequence shown in any of SEQ ID NO:2, SEQ IDNO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:21, SEQ ID NO:22, SEQ IDNO:23, SEQ ID NO:24, or SEQ ID NO:25, or a functional fragment thereof.5. The polynucleotide according to claim 1, wherein said polynucleotidecomprises the nucleotide sequence shown in any of SEQ ID NO:1, SEQ IDNO:11, SEQ ID NO:12, or SEQ ID NO:13, or a functional fragment thereof.6. (canceled)
 7. A polynucleotide encoding a chimeric plant AGPase smallsubunit protein, or a functional fragment of said protein, wherein saidchimeric AGPase protein comprises an N-terminus sequence from anN-terminus region of a plant AGPase small subunit from a first plant anda C-terminus sequence from a C-terminus region of a plant AGPase smallsubunit from a second plant, and said chimeric plant AGPase smallsubunit protein comprises an amino acid mutation wherein the amino acidcorresponding to the threonine amino acid at position 462 of wild typemaize endosperm AGPase small subunit protein is replaced by an aminoacid that confers increased heat stability when said mutant AGPase smallsubunit is expressed to form an AGPase enzyme.
 8. The polynucleotideaccording to claim 7, wherein said N-terminus sequence comprises thefirst 150 to 250 amino acids of the N-terminus region of said subunit ofAGPase of said first plant and said C-terminus sequence comprises theterminal 300 residues or less of the C-terminus region of said subunitof AGPase of said second plant.
 9. The polynucleotide according to claim7, wherein said replacement amino acid that confers increased heatstability is an isoleucine.
 10. The polynucleotide according to claim 7,wherein said N-terminus region is from maize endosperm small subunit ofAGPase and/or said C-terminus region is from potato tuber small subunitof AGPase.
 11. (canceled)
 12. The polynucleotide according to claim 7,wherein said plant AGPase small subunit protein encoded by saidpolynucleotide comprises the amino acid sequence shown in any of SEQ IDNO:4, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:10, or a functionalfragment thereof.
 13. The polynucleotide according to claim 7, whereinsaid polynucleotide comprises the nucleotide sequence shown in any ofSEQ ID NO:3, SEQ ID NO:14, SEQ ID NO:15, or SEQ ID NO:16, or afunctional fragment thereof.
 14. (canceled)
 15. A protein comprising: a)a polypeptide encoded by a polynucleotide encoding a mutant plant AGPasesmall subunit protein, or a functional fragment of said protein, saidprotein comprising an amino acid mutation wherein the amino acidcorresponding to the threonine amino acid at position 462 of wild typemaize endosperm AGPase small subunit protein is replaced by an aminoacid that confers increased heat stability when said mutant AGPase smallsubunit is expressed to form an AGPase enzyme; or b) a polypeptideencoded by a polynucleotide encoding a chimeric plant AGPase smallsubunit protein, or a functional fragment of said protein, wherein saidchimeric AGPase protein comprises an N-terminus sequence from anN-terminus region of a plant AGPase small subunit from a first plant anda C-terminus sequence from a C-terminus region of a plant AGPase smallsubunit from a second plant, and said chimeric plant AGPase smallsubunit protein comprises an amino acid mutation wherein the amino acidcorresponding to the threonine amino acid at position 462 of wild typemaize endosperm AGPase small subunit protein is replaced by an aminoacid that confers increased heat stability when said mutant AGPase smallsubunit is expressed to form an AGPase enzyme; or c) a polypeptide of a)and a polypeptide of b).
 16. A transformed or transgenic plant or planttissue or cell comprising: a) a polynucleotide encoding a mutant plantAGPase small subunit protein, or a functional fragment of said protein,said protein comprising an amino acid mutation wherein the amino acidcorresponding to the threonine amino acid at position 462 of wild typemaize endosperm AGPase small subunit protein is replaced by an aminoacid that confers increased heat stability when said mutant AGPase smallsubunit is expressed to form an AGPase enzyme; or b) a polynucleotideencoding a chimeric plant AGPase small subunit protein, or a functionalfragment of said protein, wherein said chimeric AGPase protein comprisesan N-terminus sequence from an N-terminus region of a plant AGPase smallsubunit from a first plant and a C-terminus sequence from a C-terminusregion of a plant AGPase small subunit from a second plant, and saidchimeric plant AGPase small subunit protein comprises an amino acidmutation wherein the amino acid corresponding to the threonine aminoacid at position 462 of wild type maize endosperm AGPase small subunitprotein is replaced by an amino acid that confers increased heatstability when said mutant AGPase small subunit is expressed to form anAGPase enzyme; or c) the polynucleotides of both a) and b). 17-19.(canceled)
 20. The plant or plant tissue or cell according to claim 16,wherein said plant or plant tissue or cell is monocotyledonous.
 21. Theplant or plant tissue or cell according to claim 20, wherein saidmonocotyledonous plant or plant tissue or cell is selected from thegroup consisting of rice, wheat, barley, oats, rye, sorghum, maize,sugarcane, pineapple, onion, bananas, coconut, lilies, turfgrasses, andmillet. 22-26. (canceled)
 27. A method of increasing resistance of aplant to heat stress conditions and/or increasing starch biosynthesis ofa plant, said method comprising incorporating one or more polynucleotideinto the genome of a plant and expressing the protein encoded by saidpolynucleotide, wherein said polynucleotide is or comprises: a) anypolynucleotide encoding a mutant plant AGPase small subunit protein, ora functional fragment of said protein, said protein comprising an aminoacid mutation wherein the amino acid corresponding to the threonineamino acid at position 462 of wild type maize endosperm AGPase smallsubunit protein is replaced by an amino acid that confers increased heatstability when said mutant AGPase small subunit is expressed to form anAGPase enzyme; and/or b) any polynucleotide encoding a chimeric plantAGPase small subunit protein, or a functional fragment of said protein,wherein said chimeric AGPase protein comprises an N-terminus sequencefrom an N-terminus region of a plant AGPase small subunit from a firstplant and a C-terminus sequence from a C-terminus region of a plantAGPase small subunit from a second plant, and said chimeric plant AGPasesmall subunit protein comprises an amino acid mutation wherein the aminoacid corresponding to the threonine amino acid at position 462 of wildtype maize endosperm AGPase small subunit protein is replaced by anamino acid that confers increased heat stability when said mutant AGPasesmall subunit is expressed to form an AGPase enzyme. 28-30. (canceled)31. The method according to claim 27, wherein said plant ismonocotyledonous.
 32. The method according to claim 31, wherein saidmonocotyledonous plant is selected from the group consisting of rice,wheat, barley, oats, rye, sorghum, maize, sugarcane, pineapple, onion,bananas, coconut, lilies, turfgrasses, and millet. 33-35. (canceled) 36.A method for preparing a plant having an AGPase enzyme that provides forincreased heat stability and/or increased starch biosynthesis in theplant relative to a plant having wild type AGPase enzyme, said methodcomprising introducing one or more polynucleotide into a plant cell andgrowing a plant from said plant cell, wherein said polynucleotide is orcomprises: a) any polynucleotide encoding a mutant plant AGPase smallsubunit protein, or a functional fragment of said protein, said proteincomprising an amino acid mutation wherein the amino acid correspondingto the threonine amino acid at position 462 of wild type maize endospermAGPase small subunit protein is replaced by an amino acid that confersincreased heat stabilit when said mutant AGPase small subunit isexpressed to form an AGPase enzyme; and/or b) any polynucleotideencoding a chimeric plant AGPase small subunit protein, or a functionalfragment of said protein, wherein said chimeric AGPase protein comprisesan N-terminus sequence from an N-terminus region of a plant AGPase smallsubunit from a first plant and a C-terminus sequence from a C-terminusregion of a plant AGPase small subunit from a second plant, and saidchimeric plant AGPase small subunit protein comprises an amino acidmutation wherein the amino acid corresponding to the threonine aminoacid at position 462 of wild type maize endosperm AGPase small subunitprotein is replaced by an amino acid that confers increased heatstability when said mutant AGPase small subunit is expressed to form anAGPase enzyme. 37-39. (canceled)
 40. The method according to claim 36,wherein said plant is monocotyledonous.
 41. The method according toclaim 40, wherein said monocotyledonous plant is selected from the groupconsisting of rice, wheat, barley, oats, rye, sorghum, maize, sugarcane,pineapple, onion, bananas, coconut, lilies, turfgrasses, and millet.42-44. (canceled)